Increased Expression Of IFN-γ Research Articles

The transcription factor NFAT5 inhibits experimental autoimmune encephalomyelitis (EAE) in mice

Abstract Th1 and Th17 cells contribute to the pathogenesis of multiple sclerosis (MS) and EAE, an animal model of MS. Th1 cells are characterized by the abundant expression of IFN-γ, whereas Th17 cells express high levels of IL-17. T cell activation leads to the nuclear translocation of nuclear factors and activators of T-cells (NFATs), which contribute to the expression of IFN-γ and IL-17. NFAT5 is the newest member of the NFAT family, however, its role in the pathogenesis of EAE is unknown. We are therefore studying MOG35–55-induced EAE in mice with a global heterozygous knockdown of NFAT5 (NFAT5+/−, NFAT5−/−is lethal) with NFAT5+/+ mice as a control. Interestingly, we found that the knockdown of NFAT5 actually increased EAE induction rate from 65% to 74% and delayed the recovery of EAE, indicating that NFAT5 is protective against the disease. Naïve CD4 cell differentiation reveals that the NFAT5 knockdown increased IFN-γ expression without significantly affecting IL-10 expression in Th1 cells. In vitro differentiation of Th17 cells from NFAT5+/− mice was not successful. IL-6 and protein tyrosine phosphatase SHP-1 have been demonstrated to inhibit IFN-γ expression in Th1 cells. To study the mechanisms by which NFAT5 knockdown increases IFN-γ expression in Th1 cells, we have examined the effects of NFAT5 knockdown on IL-6 and SHP-1. NFAT5 knockdown had no significant effect on IL-6 mRNA level. Instead, our ongoing experiments show that knockdown of NFAT5 reduces SHP-1 mRNA level by 38% (n=3, p=0.11) with a concomitant 44% increase in phosphorylation of Y705 of STAT3, one of its targets (n=3, p=0.2), and has no significant effect on SHP-2 mRNA level. In conclusion: NFAT5 inhibits EAE possibly by suppressing the expression of IFN-γ in Th1 cells.

Regulation of Chitinase-3-like-1 in T cell enhances anti-tumoral T cell responses to suppress lung metastasis

Abstract Chitinase-3-like-1 (Chi3l1) is known to play a significant role in the pathogenesis of Type 2 inflammation and cancer. However, the function of Chi3l1 in T cell and its clinical implications are largely unknown. Here we showed that Chi3l1 expression was increased in Th2 cells while it decreased in Th1 cells by time dependent manner. In addition, Chi3l1-deficient T cells were hyper-responsive to TcR stimulation and were prone to differentiating into Th1 cells, and retroviral transduction of Chi3l1 rescue increased IFNγ expression in Chi3l1-deficient Th1 cells. Chi3l1-deficient Th1 cells showed increased expression of anti-tumor immunity genes and decreased Th1 negative regulators. Deletion of Chi3l1 in T cells in mice showed reduced melanoma lung metastasis with increased IFNγ and TNFα-producing T cells in the lung. Furthermore, knockdown of Chi3l1 expression in the lung using peptide-siRNA complex efficiently inhibited lung metastasis with enhanced Th1 and CTL infiltration and functions. Collectively, this study demonstrates Chi3l1 is a novel regulator of Th1 and CTL which could be a therapeutic target to enhance anti-tumor immunity.

Type 1 innate lymphoid cell aggravation of atherosclerosis is mediated through TLR4.

ILC populations elaborate a similar cytokine expression pattern with helper T cell subsets Th1, Th2 and Th17. Recent studies indicate that CD25+ILC2 could alleviate atherosclerosis by altering lipid metabolism, whereas the depletion of CD90-expressing ILCs had no influence on atherosclerosis. Thus, these findings raise the question of whether ILC1 cells react on atherosclerosis. Hence, our group attempted to explore the role of ILC1 cells in atherosclerosis. We found that ILC1 cells have a high Th1-like gene expression of T-bet and IFN-γ, which is distinct from ILC2, ILC3 or conventional NK (cNK) cells. Moreover, atherosclerotic lesions were greatly reduced in ApoE-/-Rag1-/- mice treated with anti-NK1.1 mAbs for depleting ILC1 cells (ILC1+cNK cells), compared to ApoE-/-Rag1-/- mice treated with anti-IL-15R mAbs for depleting cNK cells, and these effects could be fully rescued through the adoptive transfer of ILC1 cells sorted from the spleen of ApoE-/-TLR4+/+ mice into ApoE-/-Rag1-/- mice treated with anti-NK1.1 mAbs. However, the adoptive transfer of ILC1 cells sorted from the spleen of ApoE-/-TLR4-/- mice into ApoE-/-Rag1-/- mice treated with anti-NK1.1 mAbs blocked the progression of atherosclerosis, indicating that the pro-atherosclerotic role of ILC1 cells is dependent on TLR4. Furthermore, oxLDL-induced increase in IFN-γ expression from ApoE-/- ILC1 cells was correlated with the decrease in BACH2 expression. Taken together, ILC1 cells exist in atherosclerosis and aggravate atherosclerosis via increasing pro-inflammatory cytokine expression in a TLR4/BACH2-dependent manner.

MAGE3 and Survivin activated dendritic cell immunotherapy for the treatment of non-small cell lung cancer.

Dendritic cell (DC) immunotherapy is an optimal cancer treatment, resulting in its emergence as a therapeutic choice; however, there are limited studies investigating dual antigen-pulsed DC immunotherapy in non-small cell lung cancer (NSCLC). In order to determine the effect of a recombinant melanoma-associated antigen (rMAGE-3) and recombinant Survivin (rSurvivin) peptide-pulsed DC immunotherapy in patients with NSCLC, the present clinical study was performed. DC immunotherapy was generated from the monocytes of patients with NSCLC and primed with rMAGE-3 and rSurvivin peptides. The present open-label, non-randomised study enrolled 16 patients with histologically confirmed stage I-IIIB NSCLC between December 2013 and October 2014. A prime immunotherapy (9.1×107 cells/dose) and a single boost (8.2×107 cells/dose) were administered 1 month apart intradermally and the patients were evaluated for immunological and clinical response. DC immunotherapy was well tolerated, with no serious adverse events. There was a single incidence of grade 1 fever, chills and fatigue. Out of the 16 patients enrolled, 11 patients showed stable disease and 5 showed disease progression. There was a significant increase in IFN-γ expression on day 60 vs. day 0 (P=0.048). An increasing trend in the mean cluster of differentiation (CD)4:CD8 values of day 30 and day 90 was observed, but this was not significant. The present study established that DCs primed with rMAGE-3 and rSurvivin may be used in NSCLC treatment. However, a larger study is required to address prominent issues, including secretion of immunosuppressive cytokines and mechanisms of tumour escape from immune surveillance. Several factors associated with the manufacturing and quality of immunotherapy also require standardisation.

Goblet cell loss abrogates ocular surface immune tolerance.

Intestinal epithelial cells condition tolerogenic properties in DCs. Aqueous-deficient dry eye is associated with goblet cell (GC) loss and increased IFN-γ expression in the conjunctiva. We hypothesized that loss of GCs reduces tolerance-inducing properties of antigen presenting cells (APCs) in the conjunctiva and draining nodes. Mice lacking the SAM pointed domain containing ETS transcription factor (Spdef) that is required for GC differentiation had an increased frequency of macrophages in the conjunctiva and CD11b+CD11c+ DCs in the conjunctiva and draining nodes, and these cells had greater IL-12 expression than WT mice. Conditioned media from cultured WT conjunctival GCs suppressed LPS-induced IL-12 production by conjunctival APCs. OVA antigen-specific OTII CD4+ T cells primed by Spdef-KO draining lymph node APCs showed greater proliferation, lower frequency of Foxp3+, increased frequency of IFN-γ+ and IL-17+ cells, and greater IFN-γ production than those primed by WT APCs. The immune tolerance to OVA antigen topically applied to the conjunctiva measured by cutaneous delayed type hypersensitivity (DTH) reaction, OVA-specific T cell proliferation, Foxp3 induction, and IFN-γ production observed in WT mice was lost in the Spdef-KO mice. We concluded that conjunctival GCs condition tolerogenic properties in APCs that suppress IL-12 production and Th1 polarization.

IL-18 and IFN-gamma expression enhances contact hypersensitivity after oral administration of naturally oxidized olive oil to mice

ABSTRACT The consumption of oxidized cooking oils may exacerbate some allergic diseases. A contact hypersensitivity (CHS) mouse model was used to investigate the effects of naturally oxidized olive oil on the expression of inflammatory cytokines after a sensitization and elicitation phase. The mRNA expressions of IL-12, IL-18, and IFN-γ in the ear increased seven days after sensitization. IL-18 and IFN-γ mRNA expressions after elicitation were significantly enhanced by administration of oxidized olive oil, which exacerbated ear swelling and mononuclear cell infiltration from CHS after elicitation. IL-18 expression in the ear of oxidized oil-treated mice was highest before elicitation and decreased with an increase in IFN-γ expression after elicitation. The acceleration of an immune reaction by administration of naturally oxidized olive oil in the CHS mouse model was caused by increased expression of IL-18 in the ear auricles after sensitization, which was followed by a significant increase in IFN-γ expression after elicitation.

DNA Methyltransferase Inhibition Promotes Th1 Polarization in Human CD4+CD25high FOXP3+ Regulatory T Cells but Does Not Affect Their Suppressive Capacity.

Regulatory T cells (Treg) can show plasticity whereby FOXP3 expression, the master transcription factor for Treg suppressor function, is lost and proinflammatory cytokines are produced. Optimal FOXP3 expression strongly depends on hypomethylation of the FOXP3 gene. 5-Azacytidine (Aza) and its derivative 5-aza-2′-deoxycytidine (DAC) are DNA methyltransferase inhibitors (DNMTi) that are therapeutically used in hematological malignancies, which might be an attractive strategy to promote Treg stability. Previous in vitro research primarily focused on Treg induction by DAC from naïve conventional CD4+ T cells (Tconv). Here, we examined the in vitro effect of DAC on the stability and function of FACS-sorted human naturally occurring CD4+CD25high FOXP3+ Treg. We found that in vitro activation of Treg in the presence of DAC led to a significant inhibition of Treg proliferation, but not of Tconv. Although Treg activation in the presence of DAC led to increased IFNγ expression and induction of a Thelper-1 phenotype, the Treg maintained their suppressive capacity. DAC also induced a trend towards increased IL-10 expression. In vivo studies in patients with hematological malignancies that were treated with 5-azacytidine (Vidaza) supported the in vitro findings. In conclusion, despite its potential to increase IFNγ expression, DAC does preserve the suppressor phenotype of naturally occurring Treg.

LAG-3 potentiates the survival of Mycobacterium tuberculosis in host phagocytes by modulating mitochondrial signaling in an in-vitro granuloma model.

CD4+ T-cell mediated Th1 immune responses are critical for immunity to TB. The immunomodulatory protein, lymphocyte activation gene-3 (LAG-3) decreases Th1-type immune responses in T-cells. LAG-3 expression is significantly induced in the lungs of macaques with active TB and correlates with increased bacterial burden. Overproduction of LAG-3 can greatly diminish responses and could lead to uncontrolled Mtb replication. To assess the effect of LAG-3 on the progression of Mtb infection, we developed a co-culture system wherein blood-derived macrophages are infected with Mtb and supplemented with macaque blood or lung derived CD4+ T-cells. Silencing LAG-3 signaling in macaque lung CD4+ T-cells enhanced killing of Mtb in co-cultures, accompanied by reduced mitochondrial electron transport and increased IFN-γ expression. Thus, LAG-3 may modulate adaptive immunity to Mtb infection by interfering with the mitochondrial apoptosis pathway. Better understanding this pathway could allow us to circumvent immune features that promote disease.

Immune response in bovine neosporosis: Protection or contribution to the pathogenesis of abortion.

Neospora caninum is a protozoan parasite with a preference for cattle and dogs as hosts. When N.caninum infection occurs in cattle it induces abortion, bovine neosporosis being a main cause of abortion worldwide. In dairy cattle, the economic burden of neosporosis-associated abortion is so great that it might results in closure of a farm. However, not all infected cows abort and it is not yet understood why this occurs. At present there is no effective treatment or vaccine. This review provides insights on how immune response against the parasite determines protection or contribution to abortion. Aspects on markers of risk of abortion are also discussed. Humoral immune responses are not protective against N.caninum but seropositivity and antibody level can be good markers for a diagnosis of bovine neosporosis and its associated abortion risk. In addition, humoral mechanisms against N.caninum infection and abortion differ in pure-breed and cross-breed pregnant dairy and beef cattle. Concentrations of Pregnancy Associated glycoprotein -2 (PAG-2) can also be used to predict abortion. A partially protective immune response encompasses increased IFN-γ expression, which has to be counterbalanced by other cytokines such as IL-12 and IL-10, especially towards the end of pregnancy. Although IFN-γ is required to limit parasite proliferation a critical threshold of the IFN-γ response is also required to limit adverse effects on pregnancy. In clinical terms, it may be stated that IFN-γ production and cross-breed pregnancy can protect Neospora-infected dairy cows against abortion.

Metabolites Linked to Bariatric Surgery Prolong Allograft Survival in Obese Recipients

Introduction: In transplantation, obesity has become a common co-morbidity linked to inferior transplant outcome through far-reaching effects on the complex network of metabolism, inflammation and immunity. Methods: Fully mismatched skin transplantations were performed in diet-induced obese (DIO) C57/Bl6 mice and lean littermates. Sleeve gastrectomies (SGx) were performed prior to transplantation to induce weight loss. Results: Obese graft recipients showed significantly accelerated allograft rejection when compared to lean littermates (p < 0.001), associated with increased IFN-γ expression in splenic CD4+ T cells. Bariatric surgery, on the other hand, prolonged allograft survival even beyond that of lean animals (p = 0.005) and promoted protective IL-10-skewed conditions with reduced alloreactivity as confirmed by ELISPOT. Of note, sham procedures and dietary weight loss showed inferior results in allograft survival and weight loss when compared to bariatric surgery. Quantitative metabolomic profiling identified a secondary bile acid and a proteinogenic amino acid as upregulated subsequent to bariatric surgery. Strikingly, combined application reduced obesity and resulted in significant prolongation of graft survival (p = 0.005; Fig.1). Metabolic treatment was also associated with reduced pro-inflammatory responses in splenic CD4+ T cells and a significant reduction in splenic M1-like antigen-presenting cells (Fig. 2). Mechanistically, metabolic treatment reduced M1-polarization of monocytes/macrophages through activation of the bile acid receptor TGR5. Activation of TGR5 through a specific agonist blocked M1-polarization in vitro and prolonged allograft survival to levels comparable to metabolic treatment, while disruption of intracellular signaling of TGR5 during metabolic treatment abrogated these effects. Of note, studies in metabolic cages demonstrated a reduced oral intake of treated obese animals while maintaining regular physical activity, indicating that weight loss was not induced through toxic side-effects of metabolic treatment. Conclusion: Obesity augments alloimmune responses resulting in accelerated graft rejection, while bariatric surgery induced a change in cytokine balances with transplant outcome superior even to lean controls. Two metabolic compounds were identified as key mediators skewing the interplay of metabolism and inflammation towards anti-inflammatory conditions, providing unique insights linking obesity, metabolism and immunity.FigureFigure

Splenic CD4+ T Cells in Progressive Visceral Leishmaniasis Show a Mixed Effector-Regulatory Phenotype and Impair Macrophage Effector Function through Inhibitory Receptor Expression.

Visceral leishmaniasis (VL), caused by infection with the intracellular protozoan Leishmania donovani, is a chronic progressive disease with a relentlessly increasing parasite burden in the spleen, liver and bone marrow. The disease is characterized by fever, splenomegaly, cachexia, and pancytopenia, and progresses to death if not treated. Control of Leishmania infection is mediated by Th1 (IFNγ-producing) CD4+ T cells, which activate macrophages to produce nitric oxide and kill intracellular parasites. However, despite expansion of CD4+ T cells and increased IFNγ expression in the spleen, humans with active VL do not control the infection. We used an experimental model of chronic progressive VL in hamsters, which mimics clinical and pathological features seen in humans, to better understand the mechanisms that lead to progressive disease. Transcriptional profiling of the spleen during chronic infection revealed expression of markers of both T cell activation and inhibition. CD4+ T cells isolated from the spleen during chronic progressive VL showed mixed expression of Th1 and Th2 cytokines and chemokines, and were marginally effective in controlling infection in an ex vivo T cell-macrophage co-culture system. Splenic CD4+ T cells and macrophages from hamsters with VL showed increased expression of inhibitory receptors and their ligands, respectively. Blockade of the inhibitory receptor PD-L2 led to a significant decrease in parasite burden, revealing a pathogenic role for the PD-1 pathway in chronic VL. PD-L2 blockade was associated with a dramatic reduction in expression of host arginase 1, but no change in IFNγ and inducible nitric oxide synthase. Thus, the expression of counter-regulatory molecules on splenic CD4+ T cells and macrophages promotes a more permissive macrophage phenotype and attenuates intracellular parasite control in chronic progressive VL. Host-directed adjunctive therapy targeting the PD-1 regulatory pathway may be efficacious for VL.

Transcriptome Changes Associated with Protective Immunity in T and B Cell-Deficient &amp;lt;i&amp;gt;Rag1&amp;lt;sup&amp;gt;-/-&amp;lt;/sup&amp;gt; &amp;lt;/i&amp;gt; Mutant Zebrafish

To elucidate the basis of protective immunity in T and B cell deficient rag1 -/- mutant zebrafish, we conducted microarray analysis of 15,617 genes from rag1 -/- mutant zebrafish 48 hours after a primary response and 48 hours after a secondary response. Following primary exposure, the highest fold expression differences (3.8 to 4.95) were genes for serum amyloid A, chemokine CCL-C5a (CCL-19a), signal transducer and activator of transcription (STAT) 1b, interferon regulatory factor 11, and myxovirus resistance A. Strong induction of these genes demonstrated that primary immune responses and innate immune cells were not impaired in T and B cell deficient mutant zebrafish. Following bacterial re-exposure, the highest fold expression differences (2 to 3 fold) were in chemokine CCL-C5a (CCL-19a), myomegalin, bone morphogenetic protein 4, and relaxin 3a. These genes are involved in the immune response and cell proliferation. Genes for cell receptor activation and signal transduction, cell proliferation and cytotoxic functions were also up-regulated. These findings suggest receptor activation and expansion of a cell population. Increased ifnγ expression at 48 hpi was associated with both primary and secondary immune responses.

Combination Therapy with Daratumumab and CAR-NK Targeting CS1 for Multiple Myeloma

Daratumumab (Dara), a targeted therapy utilizing a monoclonal antibody against CD38, and its combination with other are becoming a new standard of care treatment in multiple myeloma (MM). Recently, chimeric antigen receptor (CAR) T cell immunotherapy has been successful in the clinic for the treatment of leukemia and lymphoma. Our preliminary data suggest that both CS1-CAR T cells and CS1-CAR NK cells are effective in eradicating MM cells in vitro and in vivo (Chu et al., 2014, Leukemia and Chu et al., 2014, Clinical Cancer Research). In this study, we investigated the combination therapy with Dara and CS1-CAR NK cells for the treatment of relapsed MM.We first showed that that in MM patients, CD38brightCD138─CD34─CD20+CD27+ MM cancer stem-like cells (CSCs) express CS1 at levels much higher than any other cells, and are susceptible to being eradicated by CS1-CAR NK cells. However, CD34+hematopoietic stem cells from bone marrow of healthy donors do not express CS1. These data suggest that CS1-CAR NK cells can target MM CSCs, and thus may prevent relapse of MM, as ample evidence shows that relapsed or recurrent tumor cells are derived from CSCs.We also demonstrated that CD38 is highly expressed on NK and MM cells. Dara triggered IFN-γ and GZMB expression (p< 0.01) in primary human NK cells, even in the absence of crosslinking with tumor cells. Interestingly, the increase IFN-γ expression can be validated in the CD16 (+) haNK-92 (high-affinity natural killer cells), but not in the parental NK-92 cell line. Blocking the recognition between CD16 and Dara (an IgG1 mAb) with an Fc blocking Ab completely impaired Dara-induced IFN-γ and GZMB expression, indicating that Dara-induced NK cell activation is CD16-dependent. Mechanistically, Dara significantly induced phosphorylation of NFkB and STAT1, indicating that Dara induces IFN-γ and GZMB in NK cells, which may occur through CD16 and be mediated downstream by STAT1 and NFkB. We also found that Dara failed to stimulate GZMB and IFN-γ expression in CD38(-) CD16(+) NK cells, while successful in stimulating CD38(+) CD16(+) NK cells, indicating that Dara induces NK cell activation, which requires not only the binding between CD16 and Fc fragment of Dara, but also the CD38 signaling pathway. Furthermore, we found that Dara mediated cytotoxicity of NK cells against MM cells through antibody-dependent cell-mediated cytotoxicity (ADCC) against CD38-positive (e.g., MM1.S), but not CD38-negative (e.g., U266), which can be blocked by CD16 blocking Ab. Moreover, Dara displays ADCC effects in CD16(+) NK cells but not CD16(-) NK cells. When CD16(+) NK cells were armed with the CS1-CAR, ADCC is still observed against CD38(+) MM cells at low effector to target ratios, i.e., Dara still enhances cytotoxicity of CS1-CAR NK cells, which already have enhanced cytotoxicity.We observed that Dara-induced NK cell ADCC against CD38(+) MM MM1.S cells led to increased T cell proliferation and activation in a co-culture system including dendritic cells. This effect was not observed when MM U266 cells were included as the NK cell target. Out data are consistent with that recent discovery by DiLillo and Ravetch showing that engagement of monoclonal antibody can induce an antitumor vaccine effects (David J et al., Cell, 2015).To tested Dara affects NK cell survival, immunoblotting was performed with anti-cleaved Caspase-3 and anti-cleaved PARP-1 antibodies. We demonstrated that apoptotic activity was increased in both CD16(+)NK cells (primary NK and haNK-92) and parental CD16(-)NK-92 cells treated with Dara for 24 h in a dose-dependent manner. Unlike Dara’s positive effects on CD16(+) NK cells (i.e. stimulating IFN-γ production and ADCC), induction of apoptosis seems to be CD16-independent, as parental NK-92 cells, which are CD16(-), also showed an increased levels of apoptosis induced by Dara. We are testing whether the apoptosis induction is dependent on the antigen for Dara, because as mentioned above, both primary NK cells, and modified as well as unmodified NK-92 cells, that were CD38 (+).In conclusion, our study demonstrates that the combination of Dara and CS1-CAR NK cells, which target two different tumor-associated antigens, both of which have potent anti-MM efficacy, may show additive or synergistic effects; however due to the positive and negative effects of Dara on NK cells, sequential treatment rather than a concomitant treatment modality should be considered. DisclosuresNo relevant conflicts of interest to declare.

Abstract A050: Cdk5 disruption attenuates tumor PD-L1 expression via regulation of IFN-γ signaling components and promotes antitumor immunity

Abstract In order to escape normal immune surveillance, tumors mimic peripheral tissue tolerance mechanisms such as the expression of programmed cell death-ligand 1 (PD-L1), the inhibition of which can lead to potent anti-tumor responses. Here we show the contribution of cyclin dependent kinase 5 (Cdk5) to immune evasion. Cdk5 is a proline-directed serine/threonine kinase, which is highly expressed in post-mitotic neurons and directs a variety of homeostatic functions from cytoskeletal rearrangement to neurotransmitter signaling. Studies in xenograft models have linked Cdk5 activity to the generation and metabolic activity of primary tumors within the central nervous system (CNS). Here we show that Cdk5, expressed in both murine and human medulloblastoma (MB) cell lines, plays a major role in the ability of MB to avoid immune detection. First, we observed that decreased Cdk5 expression was associated with an increase in the number of infiltrating CD3+ cells in the tumor mass of clinical MB samples. Additionally, using publicly available datasets, we found that decreased Cdk5 expression is correlated with better overall survival or fewer distant metastasis in melanoma, breast cancer, glioma, and lung adenocarcinoma. Next, using a CRISPR/Cas-9 approach, we silenced Cdk5 in a murine model of Sonic Hedgehog (SHH) pathway MB. Rather than causing intrinsic growth defects in MB, interference of Cdk5 activity sensitizes tumors to killing by the normal host immune system in a CD4+ T cell-dependent manner. This rejection is associated with increased IFN-γ expression in the tumor microenvironment, as well as increased PD-L1 expression by myeloid populations in both subcutaneous and intracranial tumors. Mechanistically, we observed an attenuated response to IFN-γ stimulated expression of programmed death ligand 1 (PD-L1) on MB cells. This blunted response was recapitulated when MB cells were treated with Roscovitine, a non-selective Cdk5 inhibitor. Furthermore, using a quantitative global phosphoproteomics approach, we found that Cdk5 knockdown also increased phosphorylation of S-440 and S-443 on interferon regulatory factor binding protein 2 (IRF2BP2), an upstream co-repressor of interferon regulatory factor 2 (IRF-2). Increased phosphorylation of IRF2BP2 corresponded with stable expression of IRF-2 with a concomitant decrease in surface expression of PD-L1. These observations highlight a critical role for Cdk5 in the immune escape mechanisms of primary CNS tumors and provides new therapeutic targets for PD-1/PD-L1 directed immunotherapy. Citation Format: Rodney D. Dorand, Jr., Joseph Nthale, Jay T. Myers, Deborah S. Barkauskas, Stefanie Avril, Steven M. Chirieleison, Tej K. Pareek, Derek W. Abbott, Duncan S. Stearns, John J. Letterio, Alex Y. Huang, Agne Petrosiute. Cdk5 disruption attenuates tumor PD-L1 expression via regulation of IFN-γ signaling components and promotes antitumor immunity [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A050.

Fluorosis increases the risk of postmenopausal osteoporosis by stimulating interferon γ

Estrogen deficiency in postmenopausal women frequently activates osteoclasts (OC), accelerates bone resorption, and leads to osteoporosis (OP). Previous studies have demonstrated that interferon γ (IFNγ) could increase bone resorption and may be involved in postmenopausal OP. Fluorosis also increased the risk of fractures and dental fluorosis, and fluoride may enhance osteoclast formation and induce osteoclastic bone destruction in postmenopausal women, but the underlying mechanisms are as yet unknown. Here, we show that serum fluoride and IFNγ levels are negatively correlated with bone mineral density (BMD) in postmenopausal women residing in a fluorotic area. Estrogen suppresses IFNγ, which is elevated by fluoride, playing a pivotal role in triggering bone loss in estrogen-deficient conditions. In vitro, IFNγ is inhibited by estrogen treatment and increased by fluoride in Raw264.7 cell, an osteoclast progenitor cell line. In ovariectomized (Ovx) mice, estrogen loss and IFNγ promote OC activation and subsequent bone loss in vivo. However, IFNγ deficiency prevents bone loss in Ovx mice even in fluoride conditions. Interestingly, fluoride fails to increase IFNγ expression in estrogen receptor α (ERα)-deficient conditions, but not in ERβ-deficient conditions. These findings demonstrate that fluorosis increases the bone loss in postmenopausal OP through an IFNγ-dependent mechanism. IFNγ signaling activates OC and aggravates estrogen deficiency inducing OP. Thus, stimulation of IFNγ production is a pivotal ‘‘upstream’’ mechanism by which fluoride promotes bone loss. Suppression of IFNγ levels may constitute a therapeutic approach for preventing bone loss.

Non-Esterified Fatty Acids Profiling in Rheumatoid Arthritis: Associations with Clinical Features and Th1 Response.

ObjectivesSince lipid compounds are known to modulate the function of CD4+ T-cells and macrophages, we hypothesize that altered levels of serum non-esterified fatty acids (NEFA) may underlie rheumatoid arthritis (RA) pathogenesis.MethodsSerum levels of NEFA (palmitic, stearic, palmitoleic, oleic, linoleic, γ-linoleic, arachidonic –AA–, linolenic, eicosapentaenoic –EPA– and docosahexaenoic –DHA–) were quantified by LC-MS/MS after methyl-tert-butylether (MTBE)-extraction in 124 RA patients and 56 healthy controls (HC). CD4+ phenotype was studied by flow cytometry. TNFα, IL-8, VEGF, GM-CSF, IFNγ, IL-17, CCL2, CXCL10, leptin and resistin serum levels were quantified by immunoassays. The effect of FA on IFNγ production by PBMC was evaluated in vitro.ResultsLower levels of palmitic (p<0.0001), palmitoleic (p = 0.002), oleic (p = 0.010), arachidonic (p = 0.027), EPA (p<0.0001) and DHA (p<0.0001) were found in RA patients, some NEFA being altered at onset. Cluster analysis identified a NEFA profile (hallmarked by increased stearic and decreased EPA and DHA) overrepresented in RA patients compared to HC (p = 0.002), being associated with clinical features (RF, shared epitope and erosions), increased IFNγ expression in CD4+ T-cells (p = 0.002) and a Th1-enriched serum milieu (IFNγ, CCL2 and CXCL10, all p<0.005). In vitro assays demonstrated that imbalanced FA could underlie IFNγ production by CD4+ T-cells. Finally, changes on NEFA levels were associated with clinical response upon TNFα-blockade.ConclusionAn altered NEFA profile can be found in RA patients associated with clinical characteristics of aggressive disease and enhanced Th1 response. These results support the relevance of lipidomic studies in RA and provide a rationale for new therapeutic targets.

A pharmacogenetic study implicates SLC9a9 in multiple sclerosis disease activity.

A proportion of multiple sclerosis (MS) patients experience disease activity despite treatment. The early identification of the most effective drug is critical to impact long-term outcome and to move toward a personalized approach. The aim of the present study is to identify biomarkers for further clinical development and to yield insights into the pathophysiology of disease activity. We performed a genome-wide association study in interferon-β (IFNβ)-treated MS patients followed by validation in 3 independent cohorts. The role of the validated variant was examined in several RNA data sets, and the function of the presumed target gene was explored using an RNA interference approach in primary T cells in vitro. We found an association between rs9828519(G) and nonresponse to IFNβ (pdiscovery = 4.43 × 10(-8)) and confirmed it in a meta-analysis across 3 replication data sets (preplication = 7.78 × 10(-4)). Only 1 gene is found in the linkage disequilibrium block containing rs9828519: SLC9A9. Exploring the function of this gene, we see that SLC9A9 mRNA expression is diminished in MS subjects who are more likely to have relapses. Moreover, SLC9A9 knockdown in T cells in vitro leads an increase in expression of IFNγ, which is a proinflammatory molecule. This study identifies and validates the role of rs9828519, an intronic variant in SLC9A9, in IFNβ-treated subjects, demonstrating a successful pharmacogenetic screen in MS. Functional characterization suggests that SLC9A9, an Na(+) -H(+) exchanger found in endosomes, appears to influence the differentiation of T cells to a proinflammatory fate and may have a broader role in MS disease activity, outside of IFNβ treatment.

Varying structural environments of an ectopic epitope influence CD4+ T cell immunogenicity (APP5P.117)

Abstract Preliminary studies have indicated that epitopes adjacent to large flexible loops of antigens are often specialized for the production of interferon gamma (IFNγ). Variants of the HIV envelope glycoprotein gp120 protein were created that feature the ectopic CD4+ T-cell epitope 2W1S swapped in at two points in a variable loop (V4) of gp120. The variants (T38 and T39) were designed to place the epitope in different environments of conformational “depth” within gp120. Both antigen sequence and antigen structure contribute to CD4+ T cell immunodominance through their effects on MHCII binding and antigen processing, respectively. In order to isolate one of these two factors, antigen variants with the same ectopic CD4+ T cell epitope at different structural locations within the protein were created to eliminate the variation in MHCII binding affinity. We hypothesize that 2W1S-specific CD4+ T cells that arise from T38 immunization will display increased IFNγ expression as compared to mice immunized with T39 due to the conformationally “shallow” context of the epitope. However, any interpretation of epitope immunogenicity must take into account the global stability of the protein, and not just the local context of epitope in the native gp120. Preliminary studies indicate that T39 adopts a conformation that is more similar to that of native gp120 as compared to T38. Current studies are investigating immunogenicity of both ectopic and endogenous epitopes by multiple assays.

Effect of ?-galycoceramide as Brain Immune Modulatory in Post-weaning Social Isolation

Background Schizophrenia is a severe mental disorder characterized by breakdown of thinking process and a deficit of typical emotional responses. More recent attempts to demonstrate connections between schizophrenia and social isolation (SI) during early postnatal life. In both disorder T helper lymphocytes was loss the immune balance. The potent exogenous ligand of natural killer T cells (NKT) cells is a-galactosylceramide (α-GalCer)that can produce both T helper (Th) lymphocytes: Th1- (IFNγ) and Th2-type (IL-4, IL-10) cytokines. In this present study, we demonstrated immunomodulatory role of α-GalCer on brain cytokines of SI rats. Method Four groups of male Wistar rats were reared post weaning in social or isolated conditions for 8 weeks. After First week of weaning, α-GalCer or normal saline delivered IP every week until last week. After the last weeks, all rats were decapitated and the brains removed. Rat brain was homogenized and lysed. The levels of IFN-γ and IL-4 were determined in brain supernatant using sandwich-base ELISA kit. Results α-GalCer indicate significantly increased IFN-γ expression (p Conclusion These data suggest that levels of IFN-γ and IL-4 were regulated differently depending on the physiological circumstance. In this regard Th1 and Th2 imbalance occurred on SI condition. Moreover, α-GalCer indicated modulation between levels of IFN-γ and IL-4 in post-weaning SI.

Dynamic interaction between astrocytes and infiltrating PBMCs in context of neuroAIDS.

HIV-mediated neuropathogenesis is a multifaceted process involving several players, including resident brain cells (neurons, astrocytes, and microglia) and infiltrating cells [peripheral blood mononuclear cells (PBMCs)]. We evaluated the dynamic interaction between astrocytes and infiltrating PBMCs as it impacts HIV in the CNS. We demonstrate that human primary-derived astrocytes (PDAs) predominantly secrete Wnt 1, 2b, 3, 5b, and 10b. Wnts are small secreted glycoproteins that initiate either β-catenin-dependent or independent signal transduction. The Wnt pathway plays a vital role in the regulation of CNS activities including neurogenesis, neurotransmitter release, synaptic plasticity, and memory consolidation. We show that HIV infection of PDAs altered astrocyte Wnt profile by elevating Wnts 2b and 10b. Astrocyte conditioned media (ACM) inhibited HIV replication in PBMCs by 50%. Removal of Wnts from ACM abrogated its ability to suppress HIV replication in PBMCs. Inversely, PBMCs supernatant activated PDAs, as demonstrated by a 10-fold increase in HLA-DR and a 5-fold increase in IFNγ expression, and enhanced astrocyte susceptibility to HIV by 2-fold, which was mediated by IFNγ in a Stat-3-dependent manner. Collectively, these data demonstrate a dynamic interaction between astrocytes and PBMCs, whereby astrocyte-secreted Wnts exert an anti-HIV effect on infected PBMCs and PBMCs, in turn, secrete IFNγ that enhance astrocyte susceptibility to productive HIV infection and mediate their activation.